Anti HPV

1. NUSANTARA BIOSCIENCE ISSN: 2087-3948
Vol. 9, No. 3, pp. 282-287 E-ISSN: 2087-3956
August 2017 DOI: 10.13057/nusbiosci/n090307
Isolation of anti-idiotype minor capsid Human papillomavirus type 16
(HPV 16 L2) IgY from egg yolk as immunogen of HPV vaccine
EKA NONENG NAWANGSIH1
, SAYU PUTU YUNI PARYATI1
, JUSUF S. EFFENDY2
,
SUNARJATI SUDIGDOADI3
, EDHYANA SAHIRATMAJA4
, DIKI HILMI5
, EVI SOVIA6
1
Department of Microbiology, Faculty of Medicine, Universitas Jenderal Achmad Yani. Jl. Terusan Jendral Sudirman, PO Box 148, Cimahi 40525, West
Java, Indonesia. Tel.: +62-22-6642781, ♥
email: eka.n.nawangsih@gmail.com
2
Department Obstetric and Gynaecology, Faculty of Medicine, Universitas Padjajaran. Jatinangor, Sumedang 45363, West Java, Indonesia
3
Department of Microbiology, Faculty of Medicine, Universitas Padjajaran. Jatinangor, Sumedang 45363, West Java, Indonesia
4
Department of Biochemistry, Faculty of Medicine, Universitas Padjajaran. Jatinangor, Sumedang 45363, West Java, Indonesia
5
Depatment of Immunology, STIKES Jenderal Achmad Yani. Cimahi 40525, West Java, Indonesia
6
Department of Pharmacology, Faculty of Medicine, Universitas Jenderal Achmad Yani. Cimahi 40525, West Java, Indonesia
Manuscript received: 12 March 2017. Revision accepted: 4 July 2017.
Abstract. Nawangsih EN, Paryati SPY, Effendy JS, Sudigdoadi S, Sahiratmaja E, Hilmi D, Sovia E. 2017. Isolation of anti-idiotype
minor capsid Human papillomavirus type 16 (HPV 16 L2) IgY from egg yolk as immunogen of HPV vaccine. Nusantara Bioscience 9:
282-287. A potential strategy for the production of safe protective vaccines for human papillomavirus (HPV) infection is to utilize anti-
ids. Meanwhile, hyperimmunized hens could provide a convenient of specific immunoglobulin in their yolks (IgY). This study aimed to
isolate IgY specific to anti-idiotype HPV 16 L2 from egg yolk which could be used for future alternative immunogen for HPV vaccine.
Antibody anti-idiotype derived from chicken egg yolk that chickens immunized by HPV 16 L2 antibody. The antibodies were purified
from immunized chicken egg yolk, then electrophoresis it using SDS-PAGE method to determine the molecular weight of IgY. To
determine an amount of protein, we used fluorometer method and to confirm an existence of specific IgY using ELISA method. The
results showed that the IgY preparation dissociated into three protein major bands with molecular weights of 180; 65; and 25 kD. The
IgY and IgY specific concentration in eggs yolk increased until 8th week. After 8th week the levels decreased gradually. Samples
derived from chickens immunized showed significantly higher concentrations of IgY and IgY specifics than control (p<0.05). These
results suggested that chicken IgY could be a practical strategy in large-scale production of specific antibody anti-idiotype HPV 16 L2
for HPV Vaccine.
Keywords: Antibody anti-idiotype, egg yolk, HPV 16 L2, isolation
INTRODUCTION
Cervical cancer, the most common cancer affecting
women in developing countries, is caused by persistent
infection with “high-risk” genotypes of human
papillomaviruses (HPV) (Castellsagué 2008). Two virus-
like particles HPV VLP (virus-like-particle) vaccines
aimed to prevent infection by high-risk HPV types are
currently available: bivalent and quadrivalent vaccine (Cho
et al. 2011; Clutt et al. 2007 ). They were a recombinant
vaccine. Another potential strategy for the production of
safe protective vaccines for HPV infection is utilized anti-
ids method. This approach arose from Jerne’s idiotypic
network theory (Ladjemi 2011). The theory stated that
antigenic epitope elicits an immune response, resulting in
the production of Ab1 antibody. The ab1 antibody can, in
turn, trigger an anti-idiotypic response consisting of distinct
subsets of Ab2 antibodies: α, β, γ, and ε. The β subtype of
anti-Id Abs express the internal image of the Ag recognized
by the Ab1 Ab and can, therefore, be used as Ag surrogates
(Jerne 1974). The ab2β have successfully induced anti-
pathogen and anti-tumor B-cell and T-cell response in
several different species (Ladjemi 2012). The existing
vaccines that made of capsid HPV major (L1) can induce
high-titer antibody but type-restricted neutralizing
antibodies. Meanwhile, L2 of genital HPV types contain
broadly cross-neutralizing epitopes but low
immunogenicity (Karanaam et al. 2009). To enhance
immunogenicity IgY in eggs yolk was used as a carrier.
The laying hens are an excellent source for the large-scale
antibody production. Moreover, the phylogenetic distance
of chicken from mammals caused it can produce high-titer
antibodies against conserved mammalian antigens
compared to other experimental animals commonly used
(da Silva 2010).
This study aimed to produce IgY specific to anti-
idiotype HPV 16 L2 from egg yolk which could be used for
future alternative immunogen for HPV vaccine.
MATERIALS AND METHODS
Materials
The study used two specific pathogenic free white
leghorn hens from Bio Farma as eggs source, one hen
injected by antibody HPV 16 L2 (Gmab #2 from Santa
Cruz) and another hen injected with physiological NaCl.
Freund's adjuvant from Sigma-Aldrich used to increase

2. NAWANGSIH et al. – Isolation of antibody anti-idiotype HPV IgY 283
immunogenicity and for purification using solution A and
solution B diagnostic kit from Promega (2004).
Fluorometer reagent from Invitrogen used to measure IgY
levels and to measure IgY specific using KPL reagent.
Methods
Hen immunization
Immunization procedure based on Thermo Fisher
Scientific Inc (2012) method. The hen was immunized
subcutaneously with antibody HPV 16 L2 to generate IgY
of anti-idiotype HPV 16 L2 in eggs yolk. Booster injections
performed at week 0, 2, 4 and 5 to raise the antibody anti-
idiotype levels in eggs yolk (0.2 mg/dose/animal). For the
first injection, the antibody was emulsified in the complete
Freund's adjuvant and for three subsequent boosters in the
incomplete adjuvant. Eggs were collected daily beginning
before and after the fourth immunizations and stored at 4o
C
to be immediately purified with a diagnostic kit.
Egg Yolk preparation
Allow eggs to warm at room temperature before
starting the preparation. Break the shell carefully and drain
most ofalbumen. Transfer the yolk into a petri dish and
remove the residual albumen with a pipette and with tissue
or use the Egg Separator (Promega). Do not break the yolk
sack. When the yolk was clean, puncture the yolk sack with
a pipette tip and lets the yolk fluid drip into a tared 100 mL
beaker glass. Hold back the yolk sack with the pipette tip.
Determine the weight of the yolk to get the volume (1g = 1
mL). A yolk of an average egg was 10-15g.
Precipitation of egg by diagnostic kit
The antibodies were purified from immunized chicken
eggs yolk by A and B solution diagnostic kit from Promega
according to the established manual procedures from the
manufacturer. The antibodies were purified from
immunized chicken eggs yolk by diagnostic kit from
Promega. Place the beaker with the yolk on a magnetic
stirrer and mix for 1-2min to get a homogenous suspension.
Added three volumes of precipitation A Solution (30 mL
per 10 mL yolk) slowly and continue stirring for 5 min at
room temperature (RT). Transfer to 50 mL centrifuge tubes
and spin 10min 10000g/11500rpm at 4°C. Filter the
supernatant through a 0.45μm filter into a tared 100 mL
glass beaker and determine the weight to get the volume
(1g = 1 mL). Discard the pellet. Added a stir bar and place
on a magnetic stirrer. Added 1/3 volume Solution B
precipitation (10 mL per 30 mL) filtered supernatant and
continued stirring for 5 min at RT. Transfer to 50 mL
centrifuge tubes and spin 10 min 10000g/11500rpm at 4°C.
Discard the supernatant and dissolve the IgY pellet in 12
mL TBS by stirring on a magnetic stirrer with a small stir
bar at RT. At this point, the protein concentration will be
10 mg/mL and IgY will be ca. 75% pure. This preparation
of IgY was suitable for most applications and could be
stored at -20°C or -75°C. For further purifications of IgY,
place the centrifuge tube containing the redissolved IgY
from the first precipitation with Solution B on a magnetic
stirrer. Add 1/3 volume B solution precipitation slowly (4
mL per 12 mL redissolved pellet) and continue stirring for
5min at RT. Spin for 10min at 10000g/11500rpm 4°C.
Discard the supernatant and dissolve the IgY pellet in 3 mL
TBS by stirring on a magnetic stirrer with a small stir bar at
RT. Filter the purified IgY through a 0.2μm filter.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
Sodium dodecyl sulfate-PAGE was done under
denaturing conditions using Mini-PROTEAN II Cell
(BioRad 2014) according to the instruction of the
manufacturer. The purity of various IgY preparations was
estimated using 10% SDSPAGE, and Coomassie brilliant
blue R-250 (BioRad) was used to visualize the protein
bands. Broad-range SDS-PAGE molecular weight
standards of 15 to 225 kDa (BioRad) were used as markers.
Fluorometer
By using fluorometric detectors, sample concentrations
can be measured without the interference of contaminants
by only emitting fluorescence when bound to the specific
target molecules. Qubit fluorometer used because the Qubit
Fluorometer accurately detects very low concentrations
protein. Measurement of IgY levels according to manual
part no. MP32866 MAN0003231 Qubit®
2.0 fluorometer
from Invitrogen.
Enzyme-linked immunosorbent assay (ELISA)
The titer of IgY against HPV 16 L2 virus was measured
by an indirect noncompetitive enzyme-linked
immunosorbent assay (ELISA) according to previously
methods with modifications (Fischer and Hlinak 1996).
First, microtiter plates were coated with the catching
antibody. Monoclonal antibody HPV 16 L2 (G mab#2
Santacruz) was diluted 1: 800 in coating buffer and 50 µI
per well was added. The plates were incubated for 2 hours
at room temperature and after that overnight at 4°C. The
plates were washed three times with washing buffer (8,0 g
NaCl; 0,2 g KH2P04; 2,9 g Na2HP04 x 12 H20; 0,2 g
KCL; 0,5 mL Tween20; add 1000,0 mL Aquadest., pH
7,4). All further washing steps were carried out with this
buffer. Then plate were added blocking solution from KPL.
The plates were washed three times with washing
buffer.Samples containing the IgY were added in well
following 4-hour incubation at 37°C, the plates were
washed three times, and 50 µI per well POD-labeled anti-
chicken IgY (Abcam)were added. Plates were incubated for
3 hours at 37°C. After this time the plates were washed and
50 µL of substrate chromogen solution were pipetted into
each well. The reaction was stopped solution (50 ul/well)
after 15 min. The absorption was measured at 450 nm
using ELISA reader. The reproducibility of the experiment
was ascertained by including a blank control (PBS) and a
negative control (IgY derived from hen immunized by
NaCl) in each plate. Positive results are determined based
on three times the value of the negative control. The
substance concentration contained in the sample is
expressed by optical density. Measuring the optical density
(OD) is a common method to quantify the concentration of
substances (Beer-Lambert law), since the absorbance is

3. NUSANTARA BIOSCIENCE 9 (4): 282-287, August 2017
284
proportional to the concentration of the absorbing
substances in the sample (David W, 2001).
Data analysis
All the values will be expressed as the mean±standard
error mean and analyzed by unpaired Student’s T test. The
level of statistical significance will be set at p<0.05
RESULTS AND DISCUSSION
Nowadays, the anti-idiotypic vaccine approach has been
successfully used for various aspects of vaccinology,
especially for tumor immunotherapy (Thomas 2012).
Recently, immunoglobulins obtained from avian eggs yolk
were preferred to offer new fields of IgY application,
particularly for therapeutic and/or prophylactic use in
human and veterinary medicine. The use of chickens for
the production of polyclonal antibodies provide several
advantages over the traditional method of producing
antibodies in mammals (Schade et al. 2005). Chicken eggs
yolk as a source for antibody production represents a
reduction in animal use since chickens produce larger
amounts of antibodies than laboratory mammals. It also
possible to eliminate the collection of blood, which is
painful for the animal. The European Centre for the
Validation of Alternative Methods (ECVAM) recommends
that yolk antibodies should be used instead of mammalian
antibodies for animal welfare reasons (Schade et al. 1996).
There were several methods for purifying IgY based on
the strategy of separation of proteins from lipoproteins and
the rest of the yolk lipids. Purification methods based on
organic solvents like chloroform remain in use. Other
methods are based on affinity chromatography or on
dilution of the yolk followed by a freezing-thawing
process. Ion exchange is also often used for purification
and is usually combined with the number of salts
precipitation method e.g. polyethylene glycol (PEG),
dextran sulfate, dextran blue, sodium sulfate, ammonium
sulfate, caprylic acid or sodium citrate (Akita and Nakai
1992). Method selection was defended on the yield and
purity desired, final use of the IgY as well as material cost
and labor skills (Carlender 2002). In this study, diagnostic
kits from Promega for purifying the harvested yolk were
used, it contains 3,5% and 12% PEG .The purity level was
70-90%. The Polyethylene glycol (PEG) that used in this
process have low toxicity and widely used in
pharmaceutical production. PEG is an excellent method to
precipitate a specific protein from a complex mixture
proteins (Goldring and Coetzer 2003). In the current study,
PEG precipitation technique was found to be simple, easy
and economical (Polson 1990) for purification of
Immunoglobulins. PEG has multiple hydroxyl groups,
which become highly reactive in an aqueous phase. In the
aqueous phase, PEG acts as an anion nucleophile and can
attract positively charged substances, thereby this
electrostatic produces changes in water. Therefore, in a
PEG medium, the hydrophobic interaction of antibody
molecules are considerably enhanced which separate them
from the other proteins of the suspensions (Shafique
1996).
The IgY concentration in eggs yolk determined by
fluorometer method. By using fluorometric detectors,
sample concentrations can be measured without the
interference of contaminants by only emitting fluorescence
when bound to the specific target molecules (Qubit 2010).
The IgY concentration in eggs yolk increased during the
immunization period and the titer began to rise
dramatically in the 4th
week. Antibody levels increased
until the 8th
week, after 8th
week the levels decreased
gradually. The results were accordance with the
recommendations of Thermo Fisher Scientific Inc protocol
that the eggs collection was done at 4th
week and harvested
at 8th
week. The maximum concentration reached at the 8th
week is 6,942 ± 0,041 mg/mL and the average of 12nd
week
is 6,076 ± 0,935 mg/mL. The results were significantly
different (p<0.05) compared with the control group which
the levels were stable relatively with the average value in
12 weeks was 4,348 ± 0,167 mg/mL. The statistical test
results showed a significant difference between the
treatment group and negative control group (p<0,05).
Serum IgG antibodies of immunized chicken were
transported and accumulated in the egg yolk efficiently
(Bar-Joseph and Malkinson 1980). Previous studies
showed that isolation of IgY dengue results obtained 5,77
mg/mL (Sudjarwo et al. 2012), Other studies that isolated
antibody anti-idiotype rabies IgY obtained 9,40 mg/mL
(Paryati and Soejoedono, 2006). The concentration of
isolated antibodies was varied. The variations of antibodies
formation were influenced by several factors, including
animal age, molecular antigen size, complexity of antigenic
chemical structure, genetic constitution, the methods of
antigen insertions and doses of antigen (Leeddell and
Weeks 1995). To confirm the existence of specific IgY
against HPV, ELISA examination was performed. Specific
antibody anti-idiotype HPV 16 L2 IgY were detected
positive result at 4th week after immunization with Optical
Density value 0,575 ± 0,106. It is based on the value of the
cut-off 0,475 calculated from 3 times the average value of
the negative control. Following reimmunization, the level
of specific antibodies continued to increase at 8th
weeks
after immunization and achieve maximum value with the
value of OD 2,564 ± 0,168. After 8th week the levels
decreased gradually to reach a level of 2,051 ± 0,014 (table
2). The decreasing of IgY levels in chicken eggs is a
reflection of the loss of plasma cell populations that
produce specific antibodies. Once fully differentiated,
plasma cells die after three to six days and the resulting
level of immunoglobulin would slowly decrease due to this
process of catabolism (Tizard 2013). Meanwhile, on
chicken that is not injected antibody HPV 16 L2 (control)
showed negative results consistently. The statistical test
results showed a significant difference between the
treatment group and negative control group (p<0,05). The
pattern of increased levels of specific antibodies in
accordance with elevated levels of IgY (Table 1).

4. NAWANGSIH et al. – Isolation of antibody anti-idiotype HPV IgY 285
Table 1. Concentration of IgY in egg yolks
Week
Concentration of IgY
sample (mg /mL)
Concentration of IgY
control (mg /mL)
X ± SD X ± SD
0 4.061 ± 0.098 4.010 ± 0.099
1 4.386 ± 0.076 4.280 ± 0.071
2 4.511 ± 0.044 4.190 ± 0.099
3 4.766 ± 0.062 4.410 ± 0.099
4 5.054 ± 0.076 4.320 ± 0.035
5 6.050 ± 0.057 4.278 ± 0.088
6 6.314 ± 0.079 4.530 ± 0.071
7 6.410 ± 0.071 4.590 ± 0.085
8 6.942 ± 0.041 4.540 ± 0.057
9 6.546 ± 0.079 4.420 ± 0.085
10 6.040 ± 0.028 4.490 ± 0.099
11 5.995 ± 0.640 4.240 ± 0.071
12 5.841 ± 0.960 4.220 ± 0.099
Average 6.076 ± 0.935 4.348 ± 0.167
Figure 1. IgY titer pattern
Table 2. Optical density of antibody anti-idiotype of HPV 16 L2 IgY
Week
Optical
density (OD)
sample
Inter-
pretation
Optical
density (OD)
control
Inter-
pretation
X ± SD X ± SD
0 0.172 ± 0.056 - 0.178 ± 0.020 -
1 0.206 ± 0.089 - 0.176 ± 0.014 -
2 0.273 ± 0.107 - 0.175 ± 0.010 -
3 0.46 ± 0.106 - 0.171 ± 0.016 -
4 0.575 ± 0.106 + 0.171 ± 0.004 -
5 0.865 ± 0.104 + 0.175 ± 0.010 -
6 1.401 ±0.087 + 0.175 ± 0.011 -
7 2.158 ± 0.281 + 0.178 ± 0.010 -
8 2.564 ± 0.168 + 0.177 ± 0.011 -
9 2.254 ± 0.116 + 0.179 ± 0.015 -
10 2.142 ± 0.103 + 0.116 ± 0.011 -
11 2.137± 0.202 + 0.115 ± 0.014 -
12 2.051 ± 0.014 + 0.115 ± 0.012 -
Average 1.424 + 0.158 -
SD 0.890 0.026
Cut off
(3x control
average) 0.475
Figure 2. IgY specific for anti-idiotype HPV 16 L2 pattern
IgY and specific IgY were successfully obtained by
immunizing the hens with antibody HPV 16 L2 emulsified
in Freund’s adjuvant. The use of adjuvants to stimulate the
immune system and to enhance the immune response is
routine in antibody production. Most protein antigens,
especially small polypeptides (<10 kDa) and non-protein
antigens, usually need to be conjugated to a large
immunogenic carrier molecule to become good
immunogens. The administration of these antigens would
need an adjuvant especially if it administered in small
quantity, to assure a high quality/high quantity antibody
response by the immunized animal (Mayo 2009).
According to Schade et al. (2005) experiment, Freund’s
complete adjuvant (FCA) and Freund’s incomplete
adjuvant (FIA) remain the most effective adjuvant to
enhance antibody levels. Freund's Complete Adjuvant
(FCA) and Freund's Incomplete Adjuvant (FIA) are the
most common adjuvants used for parenteral immunization.
These adjuvants are a mixture of mineral oil, surfactant,
and heat-killed Mycobacterium tuberculosis or M. butyric
(FCA) or without mycobacteria (FIA) (Freund et al.
1937).The water-in-oil emulsion is prepared by mixing one
volume of the adjuvant (FCA or FIA) to one volume
aqueous antigen solution. In the emulsion, the antigen is
distributed in oil droplets which disperse widely after
injection in the body, hence increasing the potential for
interaction with relevant cells. Antibody production was
enhanced by Freund’s adjuvant primarily because of the
depot effect, a depot of antigen forms at the injection site
resulting sustained release of small quantities of the antigen
over a long period of time and non-specific
immunopotentiation of macrophages by surfactants and the
mycobacteria (Stills 2005). FIA is frequently used to boost
animals that received a primary antigen injection with FCA

5. NUSANTARA BIOSCIENCE 9 (4): 282-287, August 2017
286
but it also can be used as an adjuvant for primary injection.
It has adjuvant properties that favor humoral immunity
without cell-mediated-immunity, although less potently
than FCA. Our experiment used subcutaneous (s.c.)
injection method. The most common route for antigen
injection in hens is the intramuscular (i.m.) route, but the
extensive study by Swarzkoft et al. (2000) showed that the
s.c. injection method provokes higher titer than the
intramuscular (i.m.) injection.
Positive results in the ELISA examination showed
chicken’s immune response to HPV 16 L2 antibody (Ab1).
Antibodies that produced are anti-idiotype that are
expected to have the same serological characteristics with
the original antigen and can be used to replace antigens in
immunization. The ability to mimic the structure of the
original antigen (internal image) is the cornerstone of its
use as a replacement antigen. As with antigens, anti-
idiotype antibodies have the ability to bind competitively
with specific antibodies to the original antigen (Fields et al.
2002).
SDS-PAGE electrophoresis performed to illustrate the
presence of IgY in the sample based on their molecular
weight. Samples at 8th week the highest titer is checked by
electrophoresis. There were three major bands of 180, 65
and 25 kDa, and three minor bands of 100; 70 and 35 kDa.
The electrophoresis pattern was in accordance with the
standard IgY (figure 2). Narat (2003) stated that IgY had
the molecular weight greater than IgG, which is about 180
kDa or greater. ). IgY is the major low molecular weight
immunoglobulin in egg yolk (Michael 2010). The general
structure of the IgY molecule is the same as the IgG
molecule with two heavy (H) chains and two light (L)
chains. The molecular mass of the H chain in IgY is larger
than the H chain from mammals. The greater molecular
mass of IgY is due to an increased number of heavy-chain
constant domains and carbohydrate chains (Alexander et al.
2009). IgG has 3 C regions (Cγ1-Cγ3), while IgY has 4 C
regions (Cυ1-Cυ4) and the presence of one additional C
region with its two corresponding carbohydrate chains
(Charlender 2002) logically results in a greater molecular
mass of IgY compared with IgG. SDS sample buffer
containing betamercaptol divided disulfide bonds of IgY
that lead to the separation of heavy chains and light chains.
Davalos et al. (2000) stated that the IgY consists of 65-68
kDa for each H chain (heavy chain) and 25 kDa for each of
the L chain (light chain). Meanwhile band of 70 and 100
kDa suspected the Fc and Fab fragment was not assembled
into IgY yet. According to War et al. (1995), IgY Fc
fragment has a molecular weight of 76,542 kDa. Sun et al.
(2001) also stated the molecular weight of IgY Fab
fragment was 90,718 kDa. Another protein bands were
suspected the contaminant proteins.
In conclusion, the result of this study indicate that
immunization white leghorn hen with antibody HPV 16 L2
could be novel and promise strategy to produce specific
antibody anti-idiotype HPV 16 IgY in large-scale with
animal care and also could be source of low-cost antibody
anti-idiotype for HPV vaccine as immunogen
Figure 3. Proteins isolation of immunoglobulin(IgY) from egg
yolk analyzed on SDS-PAGE. Patterns of sample fractions
obtained from solution A and solution B purification
ACKNOWLEDGEMENTS
This study financially supported by Department of
Ristekdikti Indonesia. The authors gratefully acknowledge
the help given by experts and staff of the Research and
Development Department of Microbiology Faculty of
Medicine Jenderal Achmad Yani University and
Laboratory of Genetic and Biomolecular Faculty of
Medicine Padjajaran University.
REFERENCES
Akita EM, Nakai S. 1992. Immunoglobulins from egg yolk: Isolation and
purification. J Food Sci 57: 629-634.
Alexander IT, Stella MF, Brian JS, Rosaleen AC. 2009. The crystal
structure of an avian IgY-Fc fragment reveals conservation with both
mammalian IgG and IgE. Biochemistry J 48: 556-562.
Bar-Joseph M, Malkinson M. 1980. Hen egg yolk as a source of antiviral
antibodies in the enzyme-linked immunosorbent assay (ELISA): a
comparison of two plant viruses. J Virol Meth 1: 79-184.
BioRad. 2014. Guide to Polyacrylamide Gel Electrophoresis and
Detection. Bulletin 6040 Rev A US/EG. BioRad. www.bio-rad.com
Carlender D. 2002. Avian IgY antibody: in vitro and in vivo.
[Dissertation]. Faculty of Medicine Uppsala Universitet, Uppsala.
[Sweden].
Castellsagué X. 2008. Natural history and epidemiology of HPV infection
and cervical cancer. J Gynecol Oncol 110: S4-S7.
Cho HJ, Kyoung OY, Bong KY. 2011. Advances in human
papillomavirus vaccines: a patent review. Expert Opin Ther Patents
21(3): 295-309
Cutts FT, Franceschi S, Goldie S, Castellsague X, de Sanjose S, Garnett
G, Edmunds WJ, Claeys P, Goldenthal KL, Harperi DM, Markowitz
L. 2007. Human papillomavirus and HPV vaccines. Bulletin of the
World Health Organization: 85 (9). WHO, Rome.

6. NAWANGSIH et al. – Isolation of antibody anti-idiotype HPV IgY 287
Da Silva W, Tambourgi DV. 2010. IgY: A promising antibody for use in
immunodiagnostic and in immunotherapy. Vet Immunol and
Immunopathol. DOI: 10.1016/j.vetimm.2009.12.011
Davalos PL, Ortego VJL, Bastos GD, Hidalgo AR. 2000. Colloidal
stability of IgG and IgY-coated latex microspheres, colloids and
surfaces B. Biointerfaces 20: 165-175.
David W, 2001. Immunoassay Handbook, Nature Publishing Group,
London.
Fields BA, Goldbaum FA, Ysern X, Poljak RJ, Mariuzza RA. 2002.
Molecular Basis of Antigen Mimicry by An Anti idiotope. Nature
374: 739-742.
Fischer M, Hlinak A. 1996. An ELISA for the quantification of chickens
immunoglobulin (IgY) in various liquid media. ALTEX 13: 181-182
Freund J, Casals, J, Hosmer EP. 1937. Sensitization and antibody
formation after injection of tubercle bacilli and paraffin oil. Exp Biol
Med 7: 509-513.
Goldring JP, Coetzer THT. 2003. Isolation of Chicken Immunoglobulins
(IgY) from Egg Yolk. Biochem Mol Biol Edu 31: 185-187.
Jerne NK. 1974. Towards a network theory of the immune system. Ann
Immunol 125: 373-389.
Karanam B, Jagul S, Huh KW and Richard BS. 2009. Developing
vaccines against minor capsid antigen L2 to prevent papillomavirus
infection. Immunol Cell Biol 87: 287-299
Ladjemi MZ. 2012. Anti-idiotypic antibodies as cancer vaccine
achievements and future improvements. Review article. Frontier
Oncol 2 (158) : 1-4.
Leeddell E, Weeks I. 1995. Antibody Technology. In: Graham JM,
Billington D. (eds). BIOS Scientific Publisher Ltd, Oxford, UK.
Mayo SL. 2009. Non-Invasive Antibody Production. [Dissertation].
Faculty of Medicine Uppsala Universitet, Uppsala. [Sweden].
Michael A, Meenatchisundaram S, Parameswari G, Subbraj T,
Selvakumaran R, Ramalingam S. 2010. Chicken egg yolk antibodies
(IgY) as an alternative to mammalian antibodies. Indian J Sci and
Technol 3(4): 468-71
Narat M. 2003. Production of antibodies in chickens. J Food Technol.
Biotechnol: 41(3): 259-67.
Paryati SPY, Soejoedono RD. 2006. Antibodies Anti-idiotype as Vaccine
Candidate for Rabies Prevention in Animals. [Dissertation]. Bogor
Agriculture Institute, Bogor [Indonesia]
Polson, A. 1990. Isolation of IgY from the yolks of eggs by a chloroform
polyethylene glycol procedure. Immunol Invest 19: 253-8.
Promega. 2004. EGG stract IgY purification system. IgY purification
protocol. Technical Bulletin No. 188. Promega. www.promega.com.
Qubit® 2.0 Fluorometer. 2010. User manual. Catalog no. Q32866.
Manual part no. MP32866. MAN0003231
Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund
J, Terzolo HR. 2005. Chicken egg yolk antibodies (IgY-technology):
a review of progress in production and use in research and human and
veterinary medicine. Altern Lab Anim 33 (2): 129-154.
Schade R, Staak C, Hendriksen C, Erhard M, Hug H, Koch G et al. 1996.
The production of avian (egg yolk) antibodies: IgY. Alter Lab Anim
24: 925-934.
Shafique, M.K. 1996. Purification of immunoglobulins against infectious
bronchitis from egg yolk by chloroform polyethylene glycol
technique in broiler breeds. [Thesis]. University of Agriculture,
Faisalabad, Pakistan
Stills HF. 2005. Adjuvants and antibody production: dispelling the myths
associated with Freund's complete and other adjuvants. ILAR J 46(3):
280-293.
Sudjarwo SA, Sudjarwo KE, Koerniasari. 2012. Purification and
characterization protein of anti-dengue specific immunoglobulin Y
for a diagnostic kit of dengue. J Appl Pharm Sci 2 (12): 007-012.
Sun S, Mo W, Ji Y, Liu S. 2001. Preparation and mass spectrometric
study of egg yolk antibody (IgY) against rabies virus. Rapid
Commun. Mass Spectrom 15: 708-712.
Thermo Fisher Scientific Inc. 2012. Custom chicken antibody production
protocols. Thermo Fisher Scientific Inc. www.Thermofisher.com.
Thomas KE, Bejatohlah MK, Anastas P, Somdutta S, Ramachandran M
and Heinz K. 2012. The promise of the anti-idiotype concept. Review
article. Frontier Oncol 2 (196): 1-9.
Tizard IR. 2013. Veterinary Immunology. An Introduction, 9th ed. W.B.
Saunders, Philadelphia, USA.
Warr GW, Magor KE, Higgins DA. 1995. IgY: clues to the origins of
modern antibodies. Immunol Tod 16: 392-398.